Somatostatin is a neurohormone with wide variety of physiological actions. It exists in two primary molecular forms of 14 and 28 residues in length, which are both derived from the same precursor in mammalian species. The peptides require posttranslational processing for biological activation and the processing reactions determine the specificity of the peptides for targeting at least five different types of somatostatin receptors. The proposed studies will examine the molecular requirements for the posttranslational processing reactions responsible for synthesizing somatostatin 14 and 28 (SS14 and SS28). Specifically, the substrate specificity of the somatostatin processing enzyme that produces SS28 at the monobasic cleavage site, and the structural requirement for intracellular sorting of SS14 and SS28 into different secretory pathways will be examined. In the proposed studies, both wild-type human somatostatin cDNA and somatostatin cDNAs with mutations will be subcloned into the PLJ retroviral vector and expressed in a neuroendocrine cell line AtT-20 cells. Total somatostatin production will be determined by radioimmunoassay utilizing an antibody that recognizes both 5514 and 5528. The relative amounts of SS14 and SS28 will be determined by FPLC and Sephadex G-50 gel chromatography. Antisense constructs for the prohormone convertase PC3 will be subcloned into a pBamHis retroviral vector and coexpressed with somatostatin in AtT-20 cells to determine if the PC3 is a monobasic cleavage enzyme. TPA and cAMP will be used to stimulate somatostatin release from AtT-20 cells transfected with wild- type and mutant somatostatins to elucidate the structural requirements for targeting to secretory pathways.